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CBX2 and CBX7 Antagonistically Regulate Metabolic Reprogramming in Breast Cancer

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CBX2 and CBX7 Antagonistically Regulate Metabolic Reprogramming in Breast Cancer bioRxiv preprint doi: https://doi.org/10.1101/2020.08.06.239129; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. CBX2 and CBX7 antagonistically regulate metabolic reprogramming in breast cancer Mohammad Askandar Iqbal1†, Shumaila Siddiqui1, Asad Ur Rehman2#, Farid Ahmad Siddiqui3#, Prithvi Singh4#, Bhupender Kumar5 and Daman Saluja2 1Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India 2Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007 India 3Turku Centre for Biotechnology, University of Turku and Abo Akademi, Biocity, Turku 20520 Finland 4Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India 5Department of Biochemistry, Institute of Home Economics, University of Delhi, Delhi 110016, India †Correspondence to: M. A. Iqbal, Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India. Email: [email protected]. Phone: +91-11-26981717 Ext 3426 #Equal contribution Main Figures 1-6 Supplementary Figures S1-6 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.06.239129; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Striking similarity exists between metabolic changes associated with embryogenesis and tumorigenesis. Chromobox proteins-CBX2/4/6/7/8, core components of canonical polycomb repressor complex 1 (cPRC1), play essential roles in embryonic development and aberrantly expressed in breast cancer. Understanding how altered CBX expression relates to metabolic reprogramming in breast cancer may reveal vulnerabilities of therapeutic pertinence. Using transcriptomic and metabolomic data from breast cancer patients (N>3000 combined), we identified outstanding roles of CBX2 and CBX7 in positive and negative regulation of glucose metabolism, respectively. Genetic ablation experiments validated the contrasting roles of two isoforms in cancer metabolism and cell growth. Furthermore, we determined that contrary effects of CBX2 and CBX7 on breast cancer metabolism were due to differential modulation of the mTORC1 signaling by two isoforms. Underpinning the biological significance of metabolic roles, CBX2 and CBX7 were found to be the most up- and down-regulated isoforms, respectively, in breast tumors compared to normal tissues. Moreover, CBX2 and CBX7 expression (not other isoforms) correlated strongly, but oppositely, with breast tumor aggressiveness. Genomic data showed higher amplification frequency of CBX2, not CBX7, in breast tumors. Highlighting the clinical significance of findings, survival and drug sensitivity analysis revealed that CBX2 and CBX7 predicted patient outcome and sensitivity to clinical drugs. In summary, this work identifies previously unknown antagonistic roles of CBX2 and CBX7 in breast tumor metabolism, and the results presented may have implications in strategies targeting breast cancer metabolism. Significance statement Metabolic reprogramming is a hallmark of cancer. Understanding how reprogramming of metabolism is regulated in cancer may reveal therapeutically relevant targets. Using integrative approach, we elucidate the hitherto unknown antagonistic roles of CBX2 and CBX7 in breast cancer metabolism. Highlighting the relevance of identified metabolic roles, we found that CBX2 and CBX7 (not other members) are the most differentially expressed isoforms in breast tumors (compared to normals) and the only isoforms which significantly correlate with breast cancer aggressiveness. Further, CBX2/7 predicted patient outcome and response to drugs used in breast cancer treatment, underscoring clinical significance of our study. In brief, this work unravels novel metabolic roles of CBX2/7 which may have implications in breast cancer treatment. Introduction Breast cancer is a major health challenge with over 2 million cases diagnosed worldwide in 2018, second highest after lung cancer (1). In India, breast cancer has ranked number one in cancer- related deaths among women, surpassing cervical cancer (2). Although, substantial progress has been made in breast cancer treatment strategies to bring down morbidity and mortality; the patient outcome, particularly for aggressive breast cancers, remains poor. This necessitates the bioRxiv preprint doi: https://doi.org/10.1101/2020.08.06.239129; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. identification of the oncogenic mechanisms responsible for breast carcinogenesis and their evaluation for clinical relevance. Nearly a century ago, Otto Warburg observed unusual conversion of glucose into lactate by cultured cancer cells even in the presence of ample oxygen, a phenomenon known as Warburg effect or aerobic glycolysis (3). Warburg effect along with other metabolic alterations essentially constitutes metabolic reprogramming, a key adaptation by cancer cells to support their rapid proliferation (4). Metabolic reprogramming discriminates tumor cells from their normal counterparts, thus, holding immense therapeutic significance (5). Aerobic glycolysis plays a central role in channeling glucose carbons for biomass production by branching-off pathways which rely on glycolytic intermediates as substrates, thus, prioritizing anabolism over catabolism (6). Elevated lactate production by cancer cells helps conserve glucose carbons for anabolic processes rather than ATP production via oxidative phosphorylation (7). Besides, glycolysis also serves as a source of rapid ATP production in cancer cells as it does in skeletal muscle during strenuous exercise (8). Notably, FDG-PET (18Fluorodeoxyglucose-positron emission tomography) exploits addiction of cancer cells to glucose for clinical imaging of primary and secondary tumors (9). It has now become increasingly evident that benefits of glycolysis extend beyond metabolism and role of altered glycolysis has been implicated in transcriptional regulation (10), epigenetic regulation (11, 12), immune-escape (13), cell cycle (14), mitotic spindle (15), metastasis (16), and inflammation (17). Moreover, aerobic glycolysis and associated pathways contribute to chemo- and radio-resistance in cancer (18-20). Combinatorial treatments with compounds inhibiting glycolysis have shown synergy in decreasing triple-negative breast cancer cell viability (21). Taken together, aerobic glycolysis is critical for tumor growth and survival, thus, it is important to elucidate the mechanisms that contribute to its regulation in breast cancer. Metabolic changes play essential roles during embryogenesis and tumorigenesis (22, 23). For example, aerobic glycolysis facilitates biomass production during embryo development (24). Moreover, metabolic changes are crucial in determining cellular fate and differentiation (25, 26). However, contrary to embryonic development where metabolic pathways are tightly regulated, cancer cells frequently acquire deregulation in metabolic pathways through mutations and epigenetic remodelling (27). Chromobox family members-CBX2, 4, 6, 7, and 8 (collectively referred as CBX, hereafter) are conserved components crucial for the activity of canonical polycomb repressor complex (cPRC1); and play a key role in embryonic development via transcriptional repression, necessary for maintaining cellular fate decisions (28, 29). CBXs are epigenetic readers which recruit PRC1 at specific methylated histones for transcriptional repression through chromatin compaction (30). Deregulated CBX expression has been implicated in breast cancer (31-35). Recent evidence implicates PRC1 in triggering oncogenic transcriptional programs in breast cancer (36). However, the relation between CBX with metabolic reprogramming remains unclear. With this background, we conjecture that altered CBX expression may play a role in breast cancer metabolic reprogramming. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.06.239129; this version posted August 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Using the integrative approach, we attempt here to delineate the role of aberrant CBX expression in metabolic reprogramming and identify CBX2 and CBX7 (referred as CBX2/7, hereafter) as antagonistic regulators of aerobic glycolysis, acting via modulation of mTORC1 signaling in breast cancer. Further, we evaluate the biological and clinical relevance of identified metabolic roles of CBX2 and CBX7 to show that these two isoforms are most differentially expressed in breast tumors and informative about prognosis and drug sensitivity. Results Transcriptomic and metabolomic data are mutually corroborative to suggest opposing roles of CBX2 and CBX7 in breast cancer metabolism
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